Active secretion of a thermostable transglutaminase variant in Escherichia coli

Abstract

Background Streptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli. Results First, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 ℃ and 94.25% increases in melting temperature and half-life at 60 ℃ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun’s lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL. Conclusions The stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

AI and Knowledge-Based Method for Rational Design of Escherichia coli Sigma70 Promoters

“…We previously reported that cultivation at 20 °C and using a thioredoxin (TrxA) fusion tag contributed to high mTG expression levels. , The contributions of constitutive promoters for promoting soluble enzyme expression was evaluated by cultivation at 20 and 30 °C. mTG was recombinantly expressed in zymogen form carrying TrxA and the pro-region from Streptomyces hygroscopicus proH.…”

“…mTG was recombinantly expressed in zymogen form carrying TrxA and the pro-region from Streptomyces hygroscopicus proH. The bands at 48–62 kDa corresponding to the molecular weight of mTG (56.5 kDa) , displayed minor differences using different promoters of both soluble and insoluble fractions, which may be a result of previous expression optimization (Figure S5). Following cultivation at 20 °C, mTG induced by the N19 and T7 promoters displayed the highest intracellular activity, which reached 12 and 9.6 U/mL, respectively (Figure C).…”

“…We reported coexpressing mTG zymogen and TAMEP can achieve intracellular accumulation of active mTG, but coexpression of molecular chaperones was needed to promote the soluble expression of TAMEP. Here, constitutive promoters were used for inducing TAMEP expression, and expression of TrxA-proH-mTG controlled by the T7 promoter can be used in case of potential toxicity. , The mTG activity varied from 4.1–10.1 U/mL following cultivation at 20 °C, higher than the previously reported value of 9.51 U/mL (Figure E), indicating that constitutive promoters may assist soluble TAMEP expression .…”

“…We reported coexpressing mTG zymogen and TAMEP can achieve intracellular accumulation of active mTG, but coexpression of molecular chaperones was needed to promote the soluble expression of TAMEP. Here, constitutive promoters were used for inducing TAMEP expression, and expression of TrxA-proH-mTG controlled by the T7 promoter can be used in case of potential toxicity. , The mTG activity varied from 4.1–10.1 U/mL following cultivation at 20 °C, higher than the previously reported value of 9.51 U/mL (Figure E), indicating that constitutive promoters may assist soluble TAMEP expression . The mTG purified from the soluble fraction of the cell lysate showed no band with a molecular weight above that of active mTG (38 kDa), indicating that mTG was fully activated (Figure S6).…”

“…In contrast, 6 out of 10 sigma70 promoters contributed to higher mTG intracellular activity than the T7 promoter (4.3 U/mL) following cultivation at 30 °C (Figure 2D), suggesting higher cultivation temperatures can have adverse effects on soluble protein expression as previously reported. 19 We reported coexpressing mTG zymogen and TAMEP can achieve intracellular accumulation of active mTG, 17 but coexpression of molecular chaperones was needed to promote the soluble expression of TAMEP. Here, constitutive promoters were used for inducing TAMEP expression, and expression of TrxA-proH-mTG controlled by the T7 promoter can be used in case of potential toxicity.…”